You can obtain these results with just one round of PCR. Often there’s no need for nested PCR, so you get results on the first try.

How GeneRacer Works

The GeneRacer Kit ensures that only transcripts containing full-length cDNA ends are amplified. Figure 1 outlines how the GeneRacer Kit works. The advanced protocol starts at the RNA level by specifically targeting only 5' capped mRNA. In subsequent steps the cap is removed and replaced with the GeneRacer RNA Oligo. During reverse transcription, this RNA oligo sequence is incorporated into the cDNA. Only cDNA that is completely reversed transcribed will contain this known sequence. 5' RACE PCR is then performed using the homologous GeneRacer 5' Primer that is specific to the RNA oligo sequence and a gene-specific primer. The result is amplified DNA that contains the full-length 5' cDNA sequence.

Figure 1. The GeneRacer Protocol

Sensitivity and Length

To demonstrate the ability of the GeneRacer Kit to capture the full-length 5' cDNA end, we amplified the 5' ends of genes with known transcriptional start sites. Starting with total RNA and following the GeneRacer protocol we were able to amplify both long transcripts (9 kb) and rare messages present at 0.01%, or 30 copies per cell (Figure 2).

Figure 2. Amplification of a Long Transcript & Rare Message

Three and a half micrograms of HeLa total RNA was treated according to the GeneRacer protocol.

cDNA was generated with the GeneRacer Oligo dT Primer and SuperScript II RT and then PCR amplified with the GeneRacer 5' Primer and a gene-specific primer.

Genes were amplified with one round of a 35-cycle touchdown PCR protocol.

Fifteen microliters of a 50 µl PCR reaction was run on a 1.2% agarose E-Gel and visualized under UV light.

Beyond GenBank Sequence

In addition to capturing the full-length 5' end of genes with known transcriptional start sites, GeneRacer captures additional 5' end sequence of genes with unknown start sites. cDNA amplified with GeneRacer was sequenced. Those results were compared to the mRNA sequences listed in GenBank. GenBank sequences are typically derived from cDNA obtained from libraries constructed using traditional methods that often result in a loss of terminal nucleotides. As Table 1 clearly shows, the 5' cDNA ends generated with GeneRacer extend from 16 to 116 base pairs beyond that listed in GenBank.

Table 1 - Sequence Beyond Genbank

Gene

GenBank Number (mRNA)

Size (kb)

GeneRacer Sequence Beyond GenBank

Heterogeneous nuclear ribonuclear protein complex K protein (hnRNP K)

S74678

2.3

62

Subunit for coatomer complex

X70476

3.1

38

Muscle phosphofructokinase (PFKM)

M26066

2.8

16

ADP-ribosylation factor 4 (ARF4)

M36341.1

1.5

116

Isoleucil-tRNA synthetase*

U04953

4.5

49

Putative tumor suppressor (LUCA 15)

U23946.1

2.6

81

Thyroid hormone receptor coactivating protein (SMAP)

NM_006696.1

3.1

52

Cleavage and polyadenylation specificity factor

U37012.1

4.5

57

Human insulin-like growth factor 2 receptor

NM000876

9

44

* Isoleucyl-tRNA synthetase had two strong 5´ GeneRacer PCR products. Sequencing results showed that this gene produced two mRNA populations, one having a deletion, possibly an alternatively spliced intron, at the 5´ end of the mRNA.

Genes listed were amplified using the GeneRacer protocol. PCR products were gel purified using S.N.A.P. gel purification columns and cloned into the pCR4-TOPO vector (Invitrogen). For each gene, 12 clones were randomly picked and sequenced. Sequences were aligned against those in the GenBank database and sequence beyond the 5' end GenBank sequence identified. The number of additional bases beyond the first base of GenBank sequence is taken from the clone with the longest sequence for each gene.